Organic & Biomolecular Chemistry Blog

From left to right: Eric A. Appel, Anton A. A. Smith, Caitlin L. Maikawa and Gillie A. Roth

Introducing the researchers:
Eric A. Appel is an Assistant Professor of Materials Science & Engineering at Stanford University. He received a BS in Chemistry (2008) and MS in Polymer Science (2008) from Cal Poly San Luis Obispo, and a PhD in Chemistry (2013) from Cambridge University. His research group at Stanford integrates concepts and approaches from supramolecular chemistry and polymer science to develop (bio)materials that can be used as tools to better understand fundamental biological processes and to engineer advanced healthcare solutions.

Anton A. A. Smith holds a PhD from Aarhus University, Denmark. He will soon be joining the Technical University of Denmark (DTU) where he will be continuing research at the interface of chemistry and biology.

Caitlin L. Maikawa received her BASc (2016) in Chemical Engineering from the University of Toronto. She is currently working on her PhD in Bioengineering at Stanford University with Prof. Eric Appel. Her PhD research focuses on using supramolecular biomaterials to create improved insulin formulations for the treatment of diabetes.

Gillie A. Roth received her B.S. (2015) in Bioengineering from UC San Diego and then completed her PhD in Bioengineering at Stanford University with Eric Appel. Her PhD research focused on designing biomaterials to modulate the pharmacokinetics of therapeutics across diverse disease indications.

What inspired your research in this area?
We were already using a conjugate of cucurbit[7]uril with poly(ethylene glycol) (CB[7]-PEG) as a “designer” excipient in insulin formulations (https://www.nature.com/articles/s41551-020-0555-4, https://www.pnas.org/content/113/50/14189.short) to improve insulin stability and alter pharmacokinetics. In this work we figured we could exploit the affinity of CB[7] for the N-terminal phenylalanine on insulin as a tool for insulin modification. Covalent PEG conjugates of insulin had already been examined on numerous occasions in the literature, with insulin bioactivity being heavily dependent on the site of conjugation (steric repulsion from modification in the wrong spot can completely remove activity). Conjugation to the A chain N-terminal glycine significantly reduced activity, making it an interested target for stimulus responsive activation of insulin. Unfortunately, the existing means of selectively functionalizing this site are cumbersome because the preferred site of modification on insulin is the B chain N-terminal phenylalanine. The strong, selective non-covalent binding of CB[7] to this N-terminal phenylalanine presented itself as a practical shortcut to block the nucleophilicity of this site, thereby acting as a non-covalent protecting group, to allow for selective modification of the A-chain N-terminal glycine by simple acylations.

What do you personally feel is the most interesting outcome of your study?
For insulin specifically, this work enables a simple approach to selectively functionalize the A chain, which has traditionally been very challenging to modify on account of its poor nucleophilicity compared to the B chain. Our approach shortens the synthetic route towards selective conjugation at this site as the self-assembly provides direct blocking of the site in the reaction flask with simple mixing, but the dynamic non-covalent binding allows for removal of the CB[7] protection directly during purification without need for a deprotection step.

For a broader perspective, non-covalent protection groups in protein and peptide conjugation chemistry are virtually unexplored, and we show that CB-based host-guest complexation provides a simple and effective approach to blocking of aromatic amino acids to drive selective modification elsewhere on the peptide/protein. This approach is potentially applicable to selective conjugation with an array of proteins or peptides.

What directions are you planning to take with your research in future?
Insulin conjugates, and possibly stimulus responsive activation, along with excipients in formulation are areas we are very excited about. We imagine there is ample room for developing both the chemistry and clinically relevant translational research using this new approaches to non-covalent protection.

Read the full article: Site-selective modification of proteins using cucurbit[7]uril as supramolecular protection for N-terminal aromatic amino acids

See the other articles showcased in this month’s Editor’s Collection

Rory Devlin (Left) and Jonathan Sperry (Right)

Introducing the researchers:

Rory Devlin received a BSc (Hons) from The University of Auckland in 2017. Currently, he is pursuing his PhD at the same institution under the supervision of Assoc. Prof. Jonathan Sperry, exploring novel biomimetic rearrangements towards the synthesis of alkaloid natural products.

Jonathan Sperry completed his Ph.D at the University of Exeter, working on the biomimetic synthesis with Professor Chris Moody. After postdoctoral research with Dame Margaret Brimble FRS at the University of Auckland, he was appointed to a lectureship at the same institution in 2009. Jon was a Royal Society of New Zealand Rutherford Discovery Fellow from 2014-2019.

What inspired you to write this review?

Our interest in the nudicaulins was mainly from a synthetic perspective and in particular, validating the unique cascade process in their proposed biosynthesis. We were surprised nobody had written about the nudicaulins before, especially given their fascinating history.

What experimental research are you carrying out in the area?

We are using the nudicaulin structure as a lead for drug discovery – the synthetic route to the natural product is very amenable to analogue design. We are also collaborating with Professor Bernd Schneider at the Max Planck Institute for Chemical Ecology to better understand the role and distribution of the nudicaulins.

How do you hope this review will inspire future study?

Synthetic chemists’ interest in natural products is generally focused on structure and bioactivity, but this is just the tip of the iceberg – there is so much more to learn and the nudicaulins are a great example. When we choose a natural product for synthesis studies, we now aim to understand why the organism produces the compound which has has led to some great collaborations and research projects I would have never had imagined being involved in a few years ago.

Read the full article: The curious yellow colouring matter of the Iceland poppy

See the other articles showcased in this month’s Editor’s Collection

From left to right: Robin Brabham, Tessa Keenan and Martin Fascione

Introducing the researchers:

Martin Fascione received his Ph.D. from the University of Leeds in 2009, working with W. Bruce Turnbull on synthetic carbohydrate chemistry. Following a Marie Curie fellowship with Prof. Steve Withers, FRS, in Vancouver and Prof. Gideon Davies, FRS, at the University of York (2012-2014), he took up a lectureship within Chemistry at York in August 2014. His research interests include the chemical glycobiology of rare sugars, synthetic carbohydrate chemistry, and the chemical/enzymatic modification of proteins.

Robin Brabham completed his PhD studies in the Fascione group in 2020. His thesis explored the use of amber stop codon suppression as part of new methods for the incorporation of reactive aldehydes into proteins for bioconjugations.

Tessa Keenan received her PhD in 2017 from the University of York on protein O-mannosylation. Since completing her PhD, she has undertaken postdoctoral training in the Fascione group in the areas of chemical glycobiology and protein bioconjugation.

What inspired your research in this area?

We recently developed an exciting method for modifying proteins (Chem. Sci., 2018, 9, 5585-5593), using reactive a-oxo aldehydes in proteins, but a challenge of this work was extending it to any position within the protein. Periodate oxidation of vicinal diols, or amino alcohols has long been a common method to generate aldehydes in sugar and protein chemistry, it therefore seemed an obvious next step to incorporate such a motif into an unnatural amino acid.

What do you personally feel is the most interesting outcome of your study?

The ability to incorporate the a-oxo aldehyde internally within proteins is potentially very powerful for future bioconjugation studies. However, the most interesting outcome of the paper is arguably the realisation that the classical unmasking of aldehydes by sodium periodate oxidation is hindered in PBS, a very common buffer for handling proteins.

What directions are you planning to take with your research in future?

Our primary focus is the study sugars at the interface between chemistry and biology, with an emphasis on understanding the roles they play in disease and using this knowledge to develop innovative new therapeutics. To achieve this goal we have been developing new methods for synthesising sugars and modifying proteins using aldehyde handles, and are currently using this toolkit to address unanswered questions in a wide range of diseases, including prostate cancer and leishmaniasis.

Read the full article: Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation

See the other articles showcased in this month’s Editor’s Collection

Takuho Saito and Shiki Yagai

Introducing the researchers:

Takuho Saito was born in 1996 in Tochigi, Japan. He graduated Chiba University in 2019, and is currently a master’s student under the guidance of Prof. Shiki Yagai at the same University.

Shiki Yagai was born in 1975 in Japan and received his PhD in 2002 at Ritsumeikan University. Then he directly became an assistant professor at Chiba University, and became an associate professor in 2010. In July 2017, he became a full professor in Chiba University. Find out more on his lab webpage.

What inspired your research in this area?

We are always inspired by natural molecules and macromolecules to organize into intricate nanostructures, wherein non-covalent interaction such as hydrogen bonds play important role to achieve hierarchical assembly of structures.

What do you personally feel is the most interesting outcome of your study?

The answer is definitely the fact that just changing the direction of amide groups remarkably improved the thermal stability of our nano-aggregates, as prof. Hackenberger said.

What directions are you planning to take with your research in future?

We are very much interested in the introduction of more amide groups to further improve thermal stability. At the same time, we are interested in the interplay of photoisomerization of azobenzene units and supramolecular chirality.

Read the full article: Hierarchical self-assembly of an azobenzene dyad with inverted amide connection into toroidal and tubular nanostructures

See the other articles showcased in this month’s Editor’s Collection

From left to right: Kurt Gothelf , Emiliano Clo and Thorbjørn Nielsen

Introducing the researchers:

Thorbjørn Nielsen: Obtained an MSc. Degree from the Gothelf lab in 2016. He then enrolled in a joint PhD program between Novo Nordisk in Måløv and Aarhus University where he spent half of his time in each place. He graduated in 2020 and is currently a postdoc at the Gothelf lab.

Kurt Gothelf graduated in 1995 from the group of Professor K. A. Jørgensen at Aarhus University. Following a post doctoral stay in Professor M. C. Pirrung’s group at Duke University, USA, he joined the faculty at Aarhus University in 2002 as an Associate Professor. Since 2007 he has been a Full Professor working with bioconjugation, DNA nanotechnology and biosenors. Find out more on his lab webpage.

Emiliano Clo obtained his PhD in Organic Chemistry in 2006 from the Gothelf lab at Aarhus University. He took post doctoral positions from 2007-9 with Prof. Knud Jensen at University of Copenhagen and from 2009-12 in Prof. Henrik Clausen’s Copenhagen Center for Glycomics, spending the last year of which as Research Associate Professor. From 2012, Emiliano works as a Senior Research Scientist at Novo Nordisk’s Research Chemistry Unit.

What inspired your research in this area?

Bioconjugation is challenging and both at Novo Nordisk and in the Gothelf group at Aarhus University we follow closely the development of bioconjugation techniques. Itaru Hamachi’s work on directed conjugation strategies has definitely been a steady influence over the years. But, the present study is really an amalgamation of ideas from many sources.

What do you personally feel is the most interesting outcome of your study?

The most surprising observation was to learn that the conjugation pattern was identical, in spite of the varying the length of the three reagents studied.

How do you feel your research has benefitted from collaborating between industry and academia? 

It has been of key importance to the project. Thorbjørn Nielsen (who got his MSc. with Prof. Gothelf) brought the required skillset to Novo Nordisk. Novo Nordisk then provided the materials and equipment required for this project. Together we could pull all the support the projected needed: MS-MS and SPR expertise at Novo Nordisk; cell assays and FACS from Aarhus Unversity. Last, but not the least, Apigenex, Novo’s long-time CRO partners, synthesized the reagents needed.

What directions are you planning to take with your research in future? 

Concerning bioconjugation, our future aims are twofold. First, to find reactions that can label proteins quantitatively; second, to devise reagents and protocols that can be applied to more complex proteins or in more complex matrices.

Read the full article: disulphide-mediated site-directed modification of proteins

See the other articles showcased in this month’s Editor’s Collection

The Organic & Biomolecular Chemistry Editor’s collection is a showcase of some of the best articles published in the journal, hand selected by our Associate Editors and Editorial Board members. For this inaugural selection, Associate Editor Christian Hackenberger has highlighted some of his favourite recent works. Take a look at what he thought of the articles below, and find out more about the research and the researchers behind the papers in our interviews with the authors.

Christian’s Selection:

Hierarchical self-assembly of an azobenzene dyad with inverted amide connection into toroidal and tubular nanostructures

Takuho Saito and Shiki Yagai

Christian’s comments: “Small things can make a difference! Check out this very interesting paper by Yagai et al on how inverting an amide bond in foldable azobenzene dyads changes the thermal stability of self-assembled toroids and nanotubes! Amazing! This paper is part of our collection Supramolecular chemistry in OBC. ”

Find out more in our interview with the authors

Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation

Robin L. Brabham, Tessa Keenan, Annika Husken, Jacob Bilsborrow,  Ryan McBerney,  Vajinder Kumar,  W. Bruce Turnbull and Martin A. Fascione

Christian‘s comments: “Now available site-specifically! This contribution by Fascione and coworkers expands the use of Strain-Promoted Alkyne-Nitrone Cycloaddition (SPANC) ligation to modify internal α-oxo aldhehydes in proteins. Particularly remarkable is that this work describes a rare example of site-specifically incorporated aldehyde into proteins via amber stop codon suppression. Well done folks!”

Find out more in our interview with the authors

Site-selective modification of proteins using cucurbit[7]uril as supramolecular protection for N-terminal aromatic amino acids

Anton A. A. Smith, Caitlin L. Maikawa,  Gillie A. Roth and Eric A. Appel

Christian‘s comments: “A supramolecular protecting group in peptide and protein chemistry! This paper I handled myself and I agree with the reviewers that this is a nice piece of work. Appel and coworkers make clever use of cucurbit[7]uril, which blocks the nucloephilicity of an N-terminal phenylalanine. Quite handy for the selective modification of the N-terminal glycine of the A-chain in insulin!”

Find out more in our interview with the authors

Disulphide-mediated site-directed modification of proteins

Thorbjørn Nielsen,  Anders Märcher,  Zuzana Drobňáková,  Michal Hučko,  Milan Štengl,  Vojtěch Balšánek, Charlotte Wiberg,  Per F. Nielsen,  Thomas E. Nielsen, Kurt V. Gothelf and Emiliano Cló

Christian’s comments: “Gothelf and Clo from Aarhus University and Novo Nordisk report in this paper the modification of lysines in pharmaceutically relevant proteins and antibody fragments. What is special about their work is that only lysines are modified in proximity to solvent-exprosed disulfides by clever design of a bifunctional reagent, which consits of a rebridging moiety, a masked thiol and an amine reactive group. One can only wish that more of such innovative collaborations between industry and academia are reported.”

Find out more in our interview with the authors

The Curious Yellow Colouring Matter of the Iceland Poppy

Rory Devlin and Jonathan Sperry

Christian’s comments: “And finally an awesome historical overview on an intruiging yellow colored natural product, the nudicaulin. This flavoalkaloid was named by Sir Robert Robinson in 1939. Devlin and Sperry take the reader through a fascinating journey on the first structural assignment, synthesis strategies, biosynthesis and biological role. A must read not only for the natural product chemists among us!”

Find out more in our interview with the authors

Meet the Editor:

Christian P. R. Hackenberger completed his graduate studies in chemistry at the universities of Freiburg and UW Madison and his doctoral studies in 2003 at the RWTH Aachen. After a postdoctoral position at MIT, he started his own group at the Freie Universität Berlin in 2005. In 2012, he was appointed Leibniz-Humboldt Professor for Chemical Biology at the Leibniz-Research Institute for Molecular Pharmacology and the Humboldt Universität zu Berlin.

His group works on the development of new chemoselective and bioorthogonal reactions, the identification and analysis of novel PTMs, the engineering of protein-based pharmaceuticals and novel approaches to functional protein synthesis and delivery, in particular for the labeling and modification of different antibody formats. He is co-founder of the recently founded company ‘Tubulis’, which ventures into engineering better tolerable cancer drugs based on protein- and antibody-drug conjugates.

Christian is an Associate Editor for Organic & Biomolecular Chemistry since 2015 and on the Advisory board of Chemical Science and RSC Chemical Biology. His research group can be followed on Twitter @PhosphorusFive.

Outside his chemistry life he enjoys all forms of sport (Federer!), foodie (Ottolenghi!) and cultural (Theatre! Contemporary Art! Opera!) activities.